201219565 六、發明說明： 【發明所屬之技術領域】 本發明係有關於一種生物化學反應之方法，尤指一種結合反轉錄反 應與聚合酶連鎖反應(PCR)於同一裝置内進行，提升反應流程便利性之 反轉錄聚合酶連鎖反應之方法。 【先前技術】 反轉錄聚合酶連鎖反應(Reverse Transcription Polymerase Chain φ Reaction，簡稱RT-PCR)，就是應用於檢測以RNA (核糖核酸）為基底 之病毒’將訊息核糖核酸(message RNA，mRNA)，加入反轉錄反應試劑 (反轉錄酶)製造互補DNA (complementary, cDNA)，再進行聚合酶連鎖 反應’將標定的DNA (去氧核糖核酸）序列(target sequence)放大至數 百萬倍，供進行分析。 習用的反應流程為先於一試管中加入反轉錄反應試劑，再加入 RNA檢體進行反轉錄反應，之後再將反轉錄反應轉錄出的互補DNA φ 檢體加入一 PCR試管，並加入聚合酶連鎖反應所需之酵素與緩衝液， 以進行聚合酶連鎖反應。惟上述反轉錄反應與聚合酶連鎖反應的檢體 與反應物需分批分次添加至不同反應之試管内，檢體與反應物在不同 試管中的移動與暴露在空氣中的時間越長，都將造成檢體與反應物的 污染’造成反應結果失準。 再者’上述反應物有低溫冷藏之必要’以維持反應物品質之安定與 維持酵素活性。惟需低溫冷藏的運送與貯存方式，將對業者運輸以及 使用者保存上造成很大不方便’且低溫冷藏的設備也要付出較多的成 201219565 本’若貯存不當，可能造成反應物的變質，或酵素失去活性，進而影 響反應結果之準確度。 【發明内容】 為避免檢想與反應物在不同試管中移動造成污染，以及反應物在運 輸與保存過程不當造成反應物的變質，或酵素失去活性，本發明提供 實驗步驟的改良，除可確保反應結果之準確性，俾增進實驗上的便利 性與效率。 本發明係提供一種反轉錄聚合酶連鎖反應之方法，主要特徵在於備鲁 一毛細管’將反轉錄反應所需之反轉錄酶加入毛細管，經過真空濃縮 冷凍，製成一反轉錄凍乾反應劑，使用時將RNA檢體、緩衝液、聚合 酶與引子溶液加入毛細管’與反轉錄凍乾反應劑混合進行回溶，使反 轉錄反應與聚合酶連鎖反應可一併於毛細管内進行，如此將有助增加 實驗流程之便利性。 本發明之主要目的在於提供一種反轉錄聚合酶連鎖反應之方法，使 反轉錄反應所需之反應酵素可於一毛細管中製成一反轉錄凍乾反應鲁 劑’使用時將RNA檢體、緩衝液、聚合酶與引子溶液加入毛細管，藉 此使反轉錄反應與聚合酶連鎖反應無須分批分次進行，可一併於毛細 管中進行。 本發明之主要目的在於預凍乾燥程序能排除95-99%以上的水份， 使乾燥後的反轉錄凍乾反應劑能長期保存而不致變質，且因脫水徹 底、重量輕、適合常溫下長途運輸和長期保存。且反轉錄凍乾反應劑 在室溫下能穩定保存而不會影響其實驗效果。 4 201219565 本發明之次要目的在於反應物因經過預凍乾燥程序可保存較久，且 因為反轉錄凍乾反應劑貯存於毛細管内，需進行反轉錄聚合酶連鎖反 應時，可在毛細管内將反轉錄凍乾反應劑回溶後直接進行反應，減少操 作時間與避免反應混合液移動所產生的汙染。 【實施方式】 為便於說明本發明於上述發明内容一欄中所表示的中心思想，茲以 具體實施例表達。實施例中各種不同物件係按適於說明之比例、尺寸、 馨 變形量或位移量而描緣，而非按實際元件的比例予以繪製，合先敘明。 且以下的說明中，類似的元件是以相同的編號來表示。 請參閱第-圖至第三圖，本發明係提供_•種反轉錄聚合酶連鎖反應 之方法，其特徵在於： (a)備一毛細管10，並於該毛細管1〇中加入一反轉錄酶丨丨，用 以進行反轉錄反應； _ ⑻將置於毛細管1G之反轉錄酶進行-預;東乾燥程序20，該 預康乾燥程序20係包括-低溫冷;東手段與一乾燥手段，低溫冷来手段 為將具反轉錄酶U之毛細管10置入一冷康庫中，而該乾燥手段為將具 反轉錄酶U之毛細㈣進行真空抽氣之步驟，得—反轉料乾反應劑； 藉此’於毛細管10中加入一緩衝液與舰檢趙12，將該反轉錄 康乾反應劑回溶，使反轉錄反應與後續之聚合酶連鎖反應可一併於毛 細管10中進行。 明暸上述方法原理後，以下係針對本發明之動作及原理作一詳細說 201219565 請參閱第二圖與第三圖，第二圖係為預床乾燥程序2〇生產反轉錄 凍乾反應劑的主要流程’包括：⑴反轉錄酶u準備；⑵預床乾 燥程序20，（3)常溫密封保存，或後續可直接於毛細们〇中加入狀人 檢趙12、聚合酶13、緩衝液與引子溶液進行反轉錄聚合酶連鎖反應。 其中’預;東賴程序2G包括了低溫料手段與乾料段，低溫冷旅手 段又可分為—次冷來(昇華冷來)與二次冷康(解吸冷來），一次冷珠的條 件為將盛有反轉錄酶U之毛細管1G置於冷來庫中，溫度控制在就 至40C之間’預康2-4小時’目的在於使反轉錄酶^形成結晶，並使籲 多餘水分昇華；而二次冷;東的條制秘财成結晶狀之反轉錄酶u 置入冷凝H巾’溫度控制在_4G〇c至_6G«>C之間，冷凝2 4小時目的在 於進行初步乾燥。之後進行乾燥手段’以真空度低於100毫托耳 (mUhTorr)條件下進行真空抽氣錢，便完成反轉躲乾反應劑之製 備視需求可常溫密封保存，或直接於毛細管1〇中加人狀入檢趙12、 聚合酶13、緩衝_引子溶液以進行反轉錄聚合酶連鎖反應。 4續參閲第四-八圓與第E9-B圖，為反轉錄聚合酶連鎖反應之實驗參 結果，分顺f㈣反轉躲合酶連鎖反舰程·狀轉錄减乾反 應劑進行反轉錄聚合酶連鎖反應作為對照。其中反轉錄床乾反應劑 係於37C情況下儲存一星期，需使用時可用回溶緩衝液、無菌水或適 «稀釋劑復原使其回溶，並加人2 5%的甘油作麟_卜之後，加入 RNA檢趙12、聚合酶13與引子雜進行反轉縣合酶連鎖反應，第 四Α圖與第四4圓中，數字〇〜3代表由4個疑似傳染性肌肉壞死病毒 感染之水產生物個趙取得樣品的結果，S2與S3分別代表不同擴增數量 201219565 的標準質體’n代表正常水產生物核酸之陰性對照，Μ代表分子大小標 記行。 比較第四·Α ®與第四·Β圖可觀之，"的反轉錄聚合酶連鎖反應 結果與使用反轉錄凍乾反應劑進行反轉錄聚合酶連鎖反應之結果經 瘦脂凝膠電泳分析後並*會有太大差異，但後者卻能在實驗過程中提 升實驗的便利性，無論是針對反應物或緩衝液_存或運送，且反轉 錄凍乾反應劑貯存於毛細管10 (見於第三圖）内，需進行反轉錄聚合 • 酶連鎖反應時’可在毛細管10内將反轉錄練乾反應劑回溶後直接進行 反應，減少操作時間與避免反轉錄酶11移動所產生的汙染。 請參閱第五圖’本發明之第二實施態樣，與前述實施態樣之差異在 於步驟（a)之後可將聚合酶13加入具有反轉錄酶u之該毛細管1〇， 使反轉錄酶11與聚合酶13共同進行财乾燥程序2〇，後續只需加入 RNA檢艘12、反應難之_絲奸雜，並使反轉齡乾反應劑 回溶，使反轉錄反應與聚合酶連鎖反應可—併於毛細管财進行如 # 此將更便利於實驗流程之進行。 最後請參閱第六圖並配合第三圖，本發明之第三實施態樣，與前述 實施態樣之差異在於本實嶋樣巾該絲料2()之後更可進行一 預熱程序’使反轉_ 11維持·，_預餘序級遞增之溫 度梯度’其條件為先將反轉錄;東乾反應劑以·賊貯存六小時，後再將 反轉錄雜反細以26C貯存六小時，利贿段性的溫度改變，使反 轉錄凍乾反細可H冑封歸，社反轉錄糾目溫i過大導致酵 素失去作用’使反轉錄酶11保持作用活性，如此將有利於增加實驗結 201219565 果之準確性。 總結上述，本發明之方法改善了習肢轉縣合酶連鎖反應需分批 逐次加人反應物的繁ί貞’亦改善了反應物需低溫冷藏運送與貯存的不 便之處’使反應流程更加制；而毛細管1G職供了反轉錄反應與聚 合酶連鎖反應共同於其巾進行的裝置；以及該反轉料乾反應劑，可 實現反轉錄反應㈣合酶連鎖反應實麟程賴化之魏，故總結本 發明之特點如下： 1. 於毛細管10内操作之潔淨度高，減少雜菌和微粒的污染，製成 反轉錄綠反應織可直接於毛細;^ 1Q巾餅反轉錄聚合酶連鎖反 應，避免酵素與緩衝液分批逐次添加與移動所產生的汙染❶ 2. 因反轉錄凍乾反應劑在毛細管1〇内，可直接回溶後進行反轉錄 聚合酶連鎖反應，減少操作時間，增加實驗流程效率。 3. 儲存於毛細管1〇中的反轉錄凍乾反應劑能長期保存而不致變 質，且因脫水徹底、重量輕、適合常溫下長途運輸和長期保存。且反 轉錄凍乾反應劑在室溫下能穩定保存而不會影響其實驗效果。 4·反轉錄/東乾反應劑處理條件溫和，在低溫低壓下結晶乾燥；可 避免高溫高壓下反轉錄酶11的分解變性。 5.反轉錄凍乾反應劑含水量低，不易被氧化，有利於長途運輸和 長期保存，且復水性好，能迅速吸水還原成凍乾前狀態。 雖本發明是以一個最佳實施例作說明，但精於此技藝者能在不脫離 本發明精神與範疇下作各種不同形式的改變。以上所舉實施例僅用以 說明本發明而已，非用以限制本發明之範圍。舉凡不違本發明精神所 201219565 從事的種種修改或變化，俱屬本發明申請專利範圍。 【圖式簡單說明】 第一圖係本發明方法流程概念圖。 第二圖係本發明之預凍乾燥程序示意圖。 第三圖係本發明方法流程示意圖。 第四-A圖係代表習用反轉錄聚合酶連鎖反應其產物經瓊脂凝膠電 泳分析之結果示意圖。 φ 第四-B圖係代表使用反轉錄凍乾反應劑進行反轉錄聚合酶連鎖反 應其產物經瓊脂凝膠電泳分析之結果示意圖。 第五圖係本發明方法之第二實施態樣流程概念圖。 第六圖係本發明方法之第三實施態樣流程概念圖。 【主要元件符號說明】 10毛細管 13聚合酶 20預凍乾燥程序 11反轉錄酶 12RNA檢體201219565 VI. Description of the invention: [Technical field to which the invention pertains] The present invention relates to a method for biochemical reaction, in particular to a combination of a reverse transcription reaction and a polymerase chain reaction (PCR) in the same device to enhance the reaction process. A method of reverse transcription polymerase chain reaction. [Previous technique] Reverse Transcription Polymerase Chain φ Reaction (RT-PCR) is applied to detect RNA (RNA)-based virus 'message RNA (mRNA), Adding a reverse transcription reaction reagent (reverse transcriptase) to produce complementary DNA (complementary, cDNA), and then performing a polymerase chain reaction' to amplify the calibrated DNA (deoxyribonucleic acid) sequence to millions of times for analysis. The conventional reaction procedure is to add a reverse transcription reaction reagent to a test tube, and then add an RNA sample for reverse transcription reaction, and then add the complementary DNA φ sample transcribed from the reverse transcription reaction to a PCR tube and add the polymerase chain. The enzyme and buffer required for the reaction are subjected to a polymerase chain reaction. However, the above-mentioned reverse transcription reaction and the polymerase chain reaction of the sample and the reactants need to be added to the tubes of different reactions in batches, and the longer the movement of the sample and the reactants in different tubes and the time of exposure to the air, Both will cause contamination of the sample and the reactants' resulting in inaccurate reaction results. Furthermore, the above-mentioned reactants are necessary for low-temperature refrigeration to maintain the stability of the reactants and maintain the enzyme activity. However, the transportation and storage methods required for cryogenic refrigeration will cause great inconvenience to the transportation of the operators and the preservation of the users. The equipment for low-temperature refrigeration also has to pay more into 201219565. If improperly stored, it may cause deterioration of the reactants. , or the enzyme loses activity, which in turn affects the accuracy of the reaction results. SUMMARY OF THE INVENTION In order to avoid contamination of the reaction with the reactants in different test tubes, and the deterioration of the reactants caused by improper transport and preservation of the reactants, or the loss of activity of the enzyme, the present invention provides an improvement of the experimental steps, in addition to ensuring The accuracy of the reaction results, and the convenience and efficiency of the experiment are improved. The invention provides a method for reverse transcription polymerase chain reaction, and the main feature is that the preparation of a reverse transcriptase required for a reverse transcription reaction is added to a capillary tube, and is concentrated by vacuum condensation to prepare a reverse transcription lyophilization reagent. When used, the RNA sample, the buffer solution, the polymerase and the primer solution are added to the capillary and mixed with the reverse transcription lyophilization reagent to be remelted, so that the reverse transcription reaction and the polymerase chain reaction can be carried out together in the capillary, so that there will be Help increase the convenience of the experimental process. The main object of the present invention is to provide a method for reverse transcription polymerase chain reaction, which enables a reaction enzyme required for a reverse transcription reaction to be prepared into a reverse lyophilization reaction agent in a capillary tube. The liquid, the polymerase and the primer solution are added to the capillary, whereby the reverse transcription reaction and the polymerase chain reaction are carried out without batching, and can be carried out in a capillary tube. The main purpose of the invention is that the pre-freeze drying process can exclude 95-99% of the water, so that the dried reverse transcription lyophilization reagent can be preserved for a long time without deterioration, and is completely dehydrated and light in weight, and is suitable for long distance at normal temperature. Transport and long-term preservation. Moreover, the reverse transcription lyophilization reagent can be stably stored at room temperature without affecting the experimental effect. 4 201219565 The secondary objective of the present invention is that the reactants can be stored for a long time by a pre-freeze drying procedure, and because the reverse transcription lyophilization reagent is stored in a capillary tube, a reverse transcription polymerase chain reaction is required, which can be performed in a capillary tube. The reverse transcription lyophilization reagent is directly reacted after being remelted to reduce the operation time and avoid the pollution caused by the movement of the reaction mixture. [Embodiment] For the convenience of description, the central idea expressed by the present invention in the column of the above-mentioned invention is expressed by a specific embodiment. The various articles in the embodiment are drawn according to the ratio, size, amount of singularity or displacement which is suitable for the description, and are not drawn according to the proportion of the actual components, which will be described first. In the following description, like elements are denoted by the same reference numerals. Referring to the first to third figures, the present invention provides a method for the reverse transcription polymerase chain reaction, characterized in that: (a) a capillary 10 is prepared, and a reverse transcriptase is added to the capillary丨丨, used for reverse transcription reaction; _ (8) will be placed in capillary 1G reverse transcriptase - pre; east drying program 20, the pre-drying program 20 series including - low temperature cold; east means with a drying means, low temperature The cold method is to put the capillary 10 with reverse transcriptase U into a cold storage, and the drying means is a step of vacuum-extracting the capillary (4) with reverse transcriptase U to obtain a reversal dry reaction reagent. By adding a buffer to the capillary 10 and detecting it, the reverse transcription reaction is re-dissolved, so that the reverse transcription reaction and the subsequent polymerase chain reaction can be carried out in the capillary 10. After clarifying the principle of the above method, the following is a detailed description of the action and principle of the present invention. 201219565 Please refer to the second and third figures, and the second figure is the main pre-bed drying program 2 〇 production of reverse transcription lyophilization reagent The process 'includes: (1) reverse transcriptase u preparation; (2) pre-bed drying procedure 20, (3) sealed at room temperature, or can be added directly to the capillary sputum, Zhao 12, polymerase 13, buffer and primer solution Reverse transcription polymerase chain reaction was performed. Among them, 'pre-; Dong Lai program 2G includes low temperature material means and dry material section, low temperature cold travel means can be divided into - cold (sublimation cold) and secondary cold (desorption cold), once cold beads The condition is that the capillary 1G containing reverse transcriptase U is placed in a cold storage, and the temperature is controlled to be between 40 C and 'pre-constant for 2-4 hours' in order to crystallize the reverse transcriptase and make excess water Sublimation; and secondary cold; the eastern system of secret money into a crystalline reverse transcript u placed in the condensed H towel 'temperature control between _4G〇c to _6G«> C, condensation for 24 hours Perform preliminary drying. After the drying method, vacuum evacuation is carried out under the condition of a vacuum of less than 100 mTorr (mUhTorr), and the preparation of the reverse repellent reaction agent can be completed at room temperature or sealed, or directly added to the capillary. The human form was examined by Zhao 12, polymerase 13, and buffer-primer solution for reverse transcription polymerase chain reaction. 4 Continued to refer to the fourth-eight-circle and E9-B maps, which are the experimental results of the reverse transcription polymerase chain reaction, and the reverse-f (four) reverse-avoiding enzyme chain anti-ship-like transcription-reducing agent for reverse transcription The polymerase chain reaction was used as a control. The reverse transcription bed dry reaction reagent is stored in the case of 37C for one week. When it is needed, it can be reconstituted by using the re-dissolving buffer, sterile water or appropriate diluent, and adding 25% glycerol as a lining. After that, RNA detection Zhao 12, polymerase 13 and primers were added to carry out the reversed county synthase chain reaction. In the fourth and fourth rounds, the number 〇~3 represents infection by four suspected infectious muscle necrosis viruses. The result of the sample obtained by the water-producing product, S2 and S3, respectively, represents that the standard plastid of the different amplification quantity 201219565 represents a negative control of the normal water-generating nucleic acid, and Μ represents the molecular size-labeling line. Comparing the results of the fourth Α ® and the fourth Β 可 , , , , , , , , , , , , , , , , , , , , , , , , , , , , And * will be too different, but the latter can improve the convenience of the experiment during the experiment, whether it is for the reactants or buffers, or the reverse lyophilization reagent is stored in the capillary 10 (see the third In the figure, reverse transcription polymerization is required. • When the enzyme chain reaction is carried out, the reverse transcription reaction reagent can be directly dissolved in the capillary 10 to directly react, reducing the operation time and avoiding the pollution caused by the movement of the reverse transcriptase 11. Referring to the fifth embodiment, the second embodiment of the present invention differs from the foregoing embodiment in that after the step (a), the polymerase 13 can be added to the capillary 1 having the reverse transcriptase u to make the reverse transcriptase 11 Carry out the financial drying process together with the polymerase 13 2, and then only need to add the RNA to check the vessel 12, the reaction is difficult, and the reverse-aged dry reagent can be dissolved back, so that the reverse transcription reaction and the polymerase chain reaction can be - And in the capillary, such as # will be more convenient for the experimental process. Finally, referring to the sixth figure and in conjunction with the third figure, the third embodiment of the present invention differs from the foregoing embodiment in that the pre-heating program can be further performed after the silk material 2() Inversion _ 11 maintains ·, _ pre-remaining step temperature gradient 'the condition is to reverse transcription first; Donggan reagent is stored in thief for six hours, then the reverse transcript is stored at 26C for six hours, The temperature change of the bribe is so that the reverse lyophilization can be reversed, and the anti-transcriptional temperature is too large, causing the enzyme to lose its effect, so that the reverse transcriptase 11 remains active, which will help to increase the experimental knot. 201219565 The accuracy of the results. Summarizing the above, the method of the present invention improves the complex enzyme reaction of the compound to the county and needs to add the reactants in batches, and also improves the inconvenience of the reactants to be transported and stored at low temperature, so that the reaction process is further improved. The capillary 1G serves a device in which the reverse transcription reaction and the polymerase chain reaction are carried out together with the towel; and the reverse reaction dry reaction agent can realize the reverse transcription reaction (4) the enzyme chain reaction Therefore, the characteristics of the present invention are summarized as follows: 1. The cleanliness of the operation in the capillary 10 is high, and the contamination of the bacteria and particles is reduced, and the reverse transcription green reaction can be made directly to the capillary; ^ 1Q towel reverse transcription polymerase chain The reaction avoids the pollution caused by the sequential addition and movement of the enzyme and the buffer. 2. The reverse lyophilization reagent can be directly re-dissolved in the capillary 1 进行 to carry out reverse transcription polymerase chain reaction, reducing the operation time. Increase the efficiency of the experimental process. 3. The reverse transcription lyophilization reagent stored in the capillary tube can be stored for a long time without deterioration, and is completely dehydrated, light in weight, suitable for long-distance transportation at room temperature and long-term storage. Moreover, the reverse transcription lyophilization reagent can be stably stored at room temperature without affecting the experimental effect. 4. The reverse transcription/east dry reaction treatment conditions are mild, and the crystals are dried at a low temperature and a low pressure; the decomposition and degeneration of the reverse transcriptase 11 under high temperature and high pressure can be avoided. 5. Reverse transcription lyophilization reagent has low water content, is not easy to be oxidized, is conducive to long-distance transportation and long-term preservation, and has good rehydration, and can be quickly hydrated and reduced to a state before lyophilization. While the invention has been described in terms of a preferred embodiment, the embodiments of the invention may The above embodiments are merely illustrative of the invention and are not intended to limit the scope of the invention. All modifications and variations of 201219565, which are not inconsistent with the spirit of the invention, are within the scope of the invention. BRIEF DESCRIPTION OF THE DRAWINGS The first figure is a conceptual diagram of the method flow of the present invention. The second drawing is a schematic diagram of the pre-freeze drying procedure of the present invention. The third figure is a schematic flow chart of the method of the present invention. The fourth-A map represents a schematic representation of the results of agarose gel electrophoresis analysis of a product of a conventional reverse transcription polymerase chain reaction. φ The fourth-B diagram represents the results of agarose gel electrophoresis analysis using a reverse transcription lyophilization reagent for reverse transcription polymerase chain reaction. Figure 5 is a conceptual diagram of a second embodiment of the method of the present invention. Figure 6 is a conceptual diagram of a third embodiment of the method of the present invention. [Main component symbol description] 10 capillary 13 polymerase 20 pre-freeze drying program 11 reverse transcriptase 12 RNA sample